摘要
目的 探讨反义高迁移率族蛋白1(HMGB1)基因对人胰腺癌细胞的抑制作用。方法 应用分子克隆技术构建反义HMGB1基因真核表达载体pcDNA3 1/anti HMGB1,用脂质体法将其导入人胰腺癌细胞PANC 1中,经G4 18筛选获得可稳定表达反义HMGB1的人胰腺癌细胞克隆,通过逆转录聚合酶链反应、免疫印迹法和噻唑蓝比色法检测转染4 8h后胰腺癌细胞HMGB1基因表达和体外增殖活性的变化,流式细胞仪检测细胞凋亡及细胞周期情况。结果 获得了pcDNA3 1/anti HMGB1真核表达质粒,pcDNA3 1/anti HMGB1转染可使PANC 1细胞HMGB1mRNA和蛋白表达水平显著降低(P <0 .0 1)、肿瘤细胞增殖能力明显受到抑制(P <0. 0 1) ,并出现细胞周期G1期阻滞、凋亡细胞百分数增加。结论 反义HMGB1基因的表达能有效抑制胰腺癌细胞的体外增殖并促进细胞凋亡。
Objective To study the inhibitory effect of human antisense high mobility group box 1 (HMGB1) gene on the growth of human pancreatic adenocarcinoma cell line (PANC-1).Methods HMGB1 was cloned and reversely inserted into eukaryotic expression vector pcDNA3.1 to get pcDNA3.1/anti-HMGB1, which was then transfected by liposome method into PANC-1 cells.After G418 selection, HMGB1 expression of PANC-1 cells before and after transfection was detected by RT-PCR and Western blot, the cell proliferating activity in vitro was examined by MTT analysis, and cell cycle and apoptosis were assessed by flow cytometry.Results pcDNA3.1/anti-HMGB1 vector was obtained.After transfection with pcDNA3.1/anti-HMGB1, HMGB1 gene expression of PANC-1 cells was inhibited in both mRNA and protein level, and the proliferation of cultured pancreatic adenocarcinoma cells was suppressed with a suppression rate higher than 40 % on the sixth day.The cells of G1 phase increased obviously while the cell number of S phase decreased, the number of apoptosic cell increased.Conclusion Antisense HMGB1 gene can inhibit the proliferation of human ancreatic adenocarcinoma cells and increase the cell apoptosis rate.
出处
《中华普通外科杂志》
CSCD
北大核心
2005年第3期180-183,共4页
Chinese Journal of General Surgery